US7763430B2 - Diagnostic assay for anti-von Willebrand Factor cleaving protease (ADAMTS13) antibodies - Google Patents
Diagnostic assay for anti-von Willebrand Factor cleaving protease (ADAMTS13) antibodies Download PDFInfo
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- US7763430B2 US7763430B2 US10/422,052 US42205203A US7763430B2 US 7763430 B2 US7763430 B2 US 7763430B2 US 42205203 A US42205203 A US 42205203A US 7763430 B2 US7763430 B2 US 7763430B2
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
Definitions
- This invention relates to a kit to be used in an assay system for determination of an anti-von Willebrand Factor-cleaving protease (ADAMTS13) antibody (“anti-vWF-cpantibody”) in a sample suspected to comprise an anti-vWF-cp antibody.
- ADAMTS13 anti-von Willebrand Factor-cleaving protease
- the kit can be used in a method for diagnosis of disorders associated with the occurrence of anti-vWF-cp-antibodies in patients, and to discriminate between different forms of thrombotic microangiopathy.
- vWF von Willebrand Factor
- Plasma von Willebrand Factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, and especially the very large vWF multimers are haemostatically competent. The existence of plasma factors that control the size of vWF multimers has long been suspected.
- the von Willebrand Factor-cleaving protease (“vWF-cp”) is involved in the limitation of platelet thrombus growth by proteolytic cleavage of von Willebrand Factor multimers in man (Furlan et al., (1996) Blood 87: 4223-4234).
- vWFcp regulates vWF multimer size by proteolytic cleavage.
- thrombotic thrombocytopenic purpura TTP
- HUS hemolytic-uremic syndrome
- TTP Trigger relapsing TTP is associated with acquired or congenital deficiency of vWF-cp.
- the rare hereditary form of TTP has been related to specific gene mutations in the ADAMTS-13 locus.
- Acute idiopathic TTP or acquired TTP is usually more severe than chronic relapsing TTP, wherein these patients have acquired antibodies against vWF-cp, which inhibit the von Willebrand Factor-cleaving protease (Furlan et al., (1998) Blood 91: 2839-2846; Furlan et al., (1998) N. Engl. J. Med. 339: 1578-1584).
- Acquired TTP also occurs occasionally during pregnancy or in the postpartum period. Intermittent relapsing TTP is also associated with the reappearance of vWF-cp inhibitor.
- TTP For other forms of TTP, such as ticlopidine-associated TTP, it has also been observed that these patients have acquired antibodies against vWF-cp (Moake, (2002) N. Eng. J. Med. 347:589-600). However, some patients with acquired TTP having unusually large vWF multimers in plasma lack severe reduced levels of vWF-cp.
- inhibitory antibodies against proteins cause serious problems, for example within the coagulation cascade, leading to blood loss or thrombosis.
- Congenital and acquired TTP are discriminated by the presence of inhibitory antibodies against vWF-cp in the plasma of up to 80% of patients suffering from acquired TTP, and total absence of vWF-cp in plasma of patients with hereditary TTP. So far, inhibitory antibodies in plasma of patients are determined by static enzyme assays under non-physiological conditions and confirm the diagnosis of acute, antibody-mediated TTP.
- vWF-cp activity and the presence of inhibitors of vWF-cp are determined by incubation of purified vWF multimers with plasma samples of patients, followed by immunoblotting of degraded vWF substrate with anti-vWF antibodies and multimer analysis (Furlan et al., (2002) Sem. Thromb. Haemost. 28:167-172).
- the method is very sensitive in the range of low protease activity; however, the accuracy is only moderate in the subnormal or normal range of protease activity.
- a collagen-binding assay for determination vWF-cp activity and vWF-cp inhibitors as described by Gerritsen et al. [(1999) Thromb. Haemost. 82:1386-1389] can be completed within 6 hours, but the method is less sensitive in the very low range of protease activity as compared to the immunoblotting of degraded vWF multimers (Furlan et al. 2002 supra).
- the assays described in the prior art are very cumbersome, time consuming and require the expertise of laboratories familiar with the technique.
- the known prior art assays only allow for detection of vWF-cp inhibitors that impair the catalytic function of vWF-cp. Inhibitory antibodies which may impair a vWF-cp function other than the catalytic activity, e.g. endothelial cell binding, cannot be detected by these assays.
- kits for determination of an anti-vWF-cp antibody in a sample comprises vWF-cp and/or one or more vWF-cp fragment(s) immobilized on a solid phase without substantially impairing the biological property of the vWF-cp or vWF-cp-fragment(s).
- the kit of the present invention may also contain any auxiliary agents known in the art for carrying out antigen/antibody assays (e.g., ELISA, EIA, RIA etc.), such as buffer salts, buffer disclosed solutions, blocking agents, detecting agents and the like.
- the kits that are disclosed can be provided in a variety of formats, e.g., in the form of one or more containers or a microtiter plate.
- vWF-cp or a vWF-cp fragment immobilized on a solid phase provides a simple, efficient, fast and reproducible assay system for determination of the presence of an anti-vWF-cp antibody in a sample.
- vWF-cp inhibitors have been determined which were not detected in a system of the prior art.
- the kit of the present invention provides an increased sensitivity in the current assay than prior art assays and can be used to detect vWF-antibodies amounts that may be below the detection limit of known systems.
- Assays performed with the kit of present invention allows one to discriminate between anti-vWF-cp antibodies having different specificities and based on impairment of different biological functions of vWF-cp.
- the assay to be performed with the kit of the present invention further allows for a rapid diagnosis of TTP and other disorders associated with vWF-cp inhibitors, as well as differentiation of various forms of thrombotic microangiopathy (TM).
- TM thrombotic microangiopathy
- FIG. 1 shows examples of plasmids that can be used for expression of recombinant vWF-cp, vWF-cp-fragment(s), or vWF-cp or vWF-cp-fragment(s) fused to a his-tag heterologous sequence.
- FIGS. 2A and 2B show the determination of IgG ( FIG. 2A ) and IgM ( FIG. 2B ) antibodies in plasma samples of a patient versus human normal plasma.
- the error bars indicate the two times added standard deviation of normal human plasma calculated from several plasma lots.
- One aspect of the invention relates to a kit for determination of an anti-vWF-cp antibody in a sample comprising vWF-cp and/or a vWF-cp fragment immobilized on a solid phase without substantially impairing the biological property of the vWF-cp or the vWF-cp fragment.
- the vWF-cp or vWF-cp-fragment is used in the kit as diagnostic agent providing the antigenic determination site(s) capable of binding anti-vWF-cp-antibodies present in a sample.
- determination is meant to include detection, quantification and mapping of the vWF-cp antigen-binding region of an anti-vWF-cp-antibody in a sample.
- Detection means at least one positive reaction indicating the formation of an antibody/vWF-cp—or an antibody/vWF-cp fragment—complex with a detection system, e.g., a chromogenic assay.
- a sample known not to comprise any anti-vWF-antibody, e.g., normal human plasma is used as negative control.
- “Quantification” typically means that defined dilutions of a sample suspected to comprise anti-vWF-cp antibodies are contacted with the immobilized vWF-cp or a vWF-cp fragment, and the intensity of the reaction obtained by the detection system is compared to the intensity of the reaction obtained with defined dilutions of a sample comprising a known and defined amount of anti-vWF-antibodies, which is used as a standard.
- “Mapping” of the vWF-cp antigen binding site of an anti-vWF-cp antibody is performed by contacting the sample suspected to comprise anti-vWF-cp antibodies with complete vWF-cp as well as with vWF-cp fragments derived from different regions of the vWF-cp molecule. Thereby, the complete spectrum of anti-vWF-cp antibodies possibly present in a sample can be captured and anti-vWF-cp antibodies having specific binding activity within a region/domain of vWF-cp can be identified.
- sample as used herein is meant to refer to a biological fluid such as blood, plasma or tissue of a patient.
- the sample may be in particular obtained from human patients suspected of having a disorder associated with occurrence of anti-vWF-cp antibodies
- solid phase does not imply any specific limitations, and relates, for example, to an unsoluble polymer material, which can be an organic polymer, such as polyamide or a vinyl polymer (e.g., poly(meth)acrylate, polystyrene and polyvinyl alcohol, or derivates thereof), a natural polymer such as cellulose, dextrane, agarose, chitin and polyamino acids, or an inorganic polymer, such as glass or metallohydroxide.
- the solid phase can be in the form of a microcarrier, particles, membranes, strips, paper, film, pearls or plates, such as microtiter plates.
- the vWF-cp or vWF-cp fragment(s) can be immobilized on the solid phase directly by covalent coupling or via a carrier such as a linker molecule or an antibody immobilized on the solid phase.
- biological property as used herein is meant as functionally active epitopes or antigenic determinants of vWF-cp or the vWF-cp fragments, capable of binding at least one anti-vWF-cp antibody.
- the immobilization of vWF-cp or vWF-cp fragment on a solid phase is performed in such a way that the immunologic properties, in particular the structure of the functional epitopes and antigenic determinants of vWF-cp or the vWF-cp fragments are preserved and efficiently presented to be recognized by at least one anti-vWF-cp antibody present in the sample.
- the vWF-cp or vWF-cp fragments can be produced in whole or in part by recombinant techniques and can be prepared by expression in a prokaryotic or eukaryotic host system.
- Prokaryotic hosts can be bacterial cells such as E. coli or B. subtilis .
- Eukaryotic cells can be selected from the group consisting of yeast cells (e.g., Pichia strains); insect cells (e.g., Sf9, Sf 21, High Five, S2); and mammalian cells, such as MRC5, CHO, COS, 3T3, HEK 293, BHK, SK-Hep, HepG2, CV-1, and Hela.
- vectors can be used for the preparation of the vWF-cp or vWF-cp fragment(s) and can be selected from eukaryotic and prokaryotic expression vectors.
- vectors for prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc., wherein the promoters used in prokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc.
- vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, using promoters such as AOX1, GAP, GAL1, AUG1, etc; (ii) for expression in insect cells, vectors such as pMT, pAcS, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc., and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived form viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses, etc., using promoters such as CMV, SV40, EF-1 ⁇ , UbC, RSV, ADV, BP
- the vWF-cp fragment(s) can be selected from the group consisting of SEQ ID NOs1-6.
- the vWF-cp fragments(s) can be peptides exhibiting amino acid sequences contained in the vWF-cp and having preferably at least 6 amino acids, more preferably from about 6 to about 50 amino acids.
- One advantage of using said peptides as a diagnostic reagent in the present invention is the selective determination of the specificity of the anti-vWF-cp antibody.
- the peptides can be produced by standard peptide synthesis techniques.
- the vWF-cp or the vWF-cp fragment(s) are fused to a heterologous sequence.
- the heterologous sequence can be heterologous protein, polypeptide or peptide, in particular a functional peptide.
- the heterologous sequence can be a sequence having binding properties to a solid phase (e.g., the solid phase may have reactive site which allows covalent binding to the heterologous sequence, or has affinity to a carrier).
- the heterologous protein, polypeptide or peptide can be selected from the group consisting of ⁇ -galactosidase, c-myc-product, glutathione S-transferase, FLAG-tags and derivatives thereof.
- the heterologous sequence can also comprise a series of several equal or different amino acids.
- the heterologous sequence is a peptide that can form a covalent bond with the solid phase, or a polyhistidine that has high affinity, particularly to specific anti-poly-histidine antibodies.
- the heterologous sequence can be fused to vWF-cp or a vWF-cp fragment at either its N- or C-terminus.
- the heterologous sequence is typically fused to the C-terminal end of vWF-cp.
- the vWF-cp or a vWF-cp fragment is fused to the heterologous sequence such that the biological property of vWF-cp or a vWF-cp fragment is not negatively affected.
- a short peptide spacer may be inserted between the heterologous sequence and vWF-cp or a vWF-cp fragment, so as not to impede sterically the presentation of the epitopes of vWF-cp or the vWF-cp fragment.
- the vWF-cp or a vWF-cp fragment is fused to a functional affinity peptide, in particular a peptide having several histidine residues, in some instances 3 to 20 histidine residues, and in other instances 6 to 15 histidine residues.
- a functional affinity peptide in particular a peptide having several histidine residues, in some instances 3 to 20 histidine residues, and in other instances 6 to 15 histidine residues.
- His-tag poly-histidine
- the immobilization on the solid phase can be effected (e.g., directly or by covalent binding) via reactive groups of the solid phase and the heterologous sequence, or via a carrier having affinity to the heterologous sequence.
- the heterologous sequence has high affinity to a carrier and the vWF-cp or vWF-cp fragment(s) are immobilized on the solid phase via the binding of its heterologous part to the carrier. Accordingly, the heterologous sequence has specific binding properties or high affinity to the carrier.
- the carrier is an antibody having affinity to the heterologous part of the vWF-cp fusion protein.
- vWF-cp or a vWF-cp fragment is fused to a poly histidine-tag as heterologous sequence and an anti-his-tag antibody is used as a carrier to immobilize vWF-cp or the vWF-cp fragment on a solid phase.
- Other heterologous affinity peptides and respective anti-affinity-peptide antibodies known to the person skilled in the art can also be used to immobilize the vWF-cp or vWF-cp fragment fusion protein.
- vWF-cp and/or vWF-cp fragment(s), or fusion proteins thereof are immobilized on the solid phase separately on different spots, e.g. in different wells of a microtiter plate, wherein typically one defined antigen such as vWF-cp or a specific vWF-fragment is contained in one spot.
- typically one defined antigen such as vWF-cp or a specific vWF-fragment is contained in one spot.
- anti-vWF-cp-antibodies can be selectively removed from the plasma of a patient identified to have specific anti-vWF-cp antibodies by subjecting the patient's plasma to affinity chromatography such as described herein which uses as an adsorbent specific vWF-cp fragments used in the assay and which have affinity to the antibody or antibodies. This allows for an improved treatment of patients having disorders associated with anti-vWF-cp antibodies compared to prior art methods.
- the kit as described above further comprises as diagnostic agent an anti-vWF-cp antibody immobilized on the solid phase.
- the anti-vWF-cp antibody can be a monoclonal antibody derived by conventional hybridoma techniques or can be an antibody or antibody fragment obtained by recombinant technique, e.g., phage display or ribosome display.
- phage display or ribosome display Such a set up in the kit of the present invention allows for differential diagnosis of thrombotic microangiopathic disorders.
- kits comprising immobilized vWF-cp, vWF-cp fragment(s) and anti-vWF-cp antibody on a solid phase the presence/absence of anti-vWF antibodies as well as the presence/absence of vWF-cp in a sample can be determined with one simple test system.
- the present invention is also related to a method for determination of an anti-vWF-cp antibody in a sample, comprising the steps of providing vWF-cp and/or one or more vWF-cp fragment(s) immobilized on a solid phase without substantially impairing the biological property of the vWF-cp or vWF-cp fragment(s), contacting a biological sample of a patient suspected of having a disorder associated with the occurrence of anti-vWF-cp antibody with the immobilized vWF-cp and/or one or more vWF-cp fragments, and detecting a complex of anti-vWF-cp antibody/vWF-cp and/or anti-vWF-cp antibody/vWF-cp fragment(s).
- the complex of anti-vWF-cp antibody/vWF-cp or anti-vWF-cp antibody/vWF-cp fragment(s) can be detected by methods well known in the art, e.g. by detection with a labelled antibody.
- the detection method can be selected from the group consisting of an enzyme assay, a chromogenic assay, a lumino assay, a fluorogenic assay, and a radioimmune assay.
- the reaction conditions to perform detection of the antibody/antigen-/complex formation depends upon the detection method selected. It is within the knowledge of the person skilled in the art to choose the optimal parameters, such as buffer system, temperature and pH for the respective detection system to be used.
- the invention also relates to a method for differential diagnosis of thrombotic microangiopathic disorders with a kit as described above, wherein the kit comprises as diagnostic agent(s) either vWF-cp and/or one or more vWF-fragments, or vWF-cp and/or vWF-fragments and anti-vWF-cp antibodies, immobilized on a solid phase.
- the diagnostic agents are preferably each located on separate spots on the solid phase, e.g. in separate wells of a microtiter plate. This allows one to differentiate between samples comprising either vWF-cp or anti-vWF-cp antibodies or both by one assay system and to differentiate between thrombotic microangiopathic disorders, e.g. different forms of TTP or HUS.
- kit and method of the present invention can be used for diagnosis of a disorder associated with occurrence of anti-vWF-cp antibodies.
- the kit and method of the present invention of the invention can also be used for diagnosis of different forms or disorders of thrombotic microangiopathy.
- the thrombotic microangiopathic (TM) disorder can be thrombotic thrombocytic purpura (TTP), neonatal thrombocytopenia, Henoch-Schönlein purpura, preclampsia, or hemolytic—uremic syndrome (HUS), HELLP syndrome, ARDS, peripheral digit ischemic syndrome, nonocclusive mesenteric ischemia, acute pancreatitis, acute hepatitis, purpura rheumatica, medicament-associated formation of thrombocytopenia, post-operative TM, cancer-associated TM, disseminated intravascular coagulation (DIC), systemic lupus erythematosus, liver cirrhosis, uremia, or acute inflammatory disorders.
- TTP thrombotic thrombocytic purpura
- HUS hemolytic—uremic syndrome
- kits and a method of the present invention This allows the fast and sensitive diagnosis of TTP and urgent needed life-saving clinical intervention, i.e. plasma treatment.
- the kit and the method of the present invention can be used for the differential diagnosis of various forms of TTP.
- kits and the method of the present invention all IgG classes as well as IgM antibodies can be detected, whereas prior art methods only allow detection of anti-vWF-cp antibodies of the IgG class.
- vWF-cp protein For expression of vWF-cp protein the vWF-cp cDNA clone as described in WO 02/42442 is used.
- vWF-cp his-tag fusion two consecutive PCRs are carried out to add the codons for 3 ⁇ glycine, 6 ⁇ histidines, stop and a XhoI restriction site.
- PCR1 the wild-type full length pcDNA3.1.(+)/vWF-cp (ADAMTS13) as described in WO 02/42441 is used as template.
- primers 7189 5′ GTG ATG GTG ATG GTG TCC ACC TCC GGT TCC TTC CTT TCC CTT CCA3′
- 6526 5′ CTG CCT CGC CCG GAA CCC CA 3′
- a 1.3 kb fragment encompassing the C-terminal SgrAI/XhoI fragment from pcDNA3.1.(+)/vWF-cp is amplified.
- the second PCR is performed.
- the resulting product is purified, digested with SgrAI and XhoI, and used to replace the corresponding SgrAI/XhoI fragment in pcDNA3.1.(+)/vWF-cp wild-type construct.
- vWF-cp fragment constructs containing different fragments of the gene of the mature protein are generated by PCR using the following primer combinations (see also Table 4 of Primers and respective vWF-cp domain sequences).
- E. coli Expression System pBAD/Topo Thiofusion (Invitrogen)
- PCR fragments are cut with suitable restriction enzymes and cloned into the vector such as pRSET ( FIG. 1 ), and cleaved with the same enzymes resulting in the desired plasmids.
- vWF-cp fragment(s)-his tag fusions For construction of vWF-cp fragment(s)-his tag fusions, the vWF-cp fragments are modified according to construction of vWF-cp/his-tag as described above.
- the constructs are cloned with HIS-6 tag by substitution of the Ndel-Xhol fragment by the synthetic oligonucleotides o.pRET-FPdHIS(1)-6929 and o.pRSET-FPdHIS(2)-6930 ( FIG. 1 ).
- vWF-cp, vWF-cp fragments or the respective his-tag fusions are recombinantly expressed in E. coli JM 109, purified and used for immobilization on a solid phase as described below.
- HEK 293 (ATCC) cells are co-transfected with pcDNA3.1.(+)/vWF-cp/C-His and a selection plasmid harboring the hygromycine cassette using calcium phosphate precipitation.
- Initial clones and subsequent subclones are selected in culture medium supplemented with 100 ⁇ g/ml hygromycine and 800 ⁇ g/ml G418 (neomycinphosphotransferase encoded on pcDNA).
- Recombinant expressed vWF-cp/his—tag is purified and used for immobilization on a solid phase as described below.
- vWF-cp-His-tag or vWF-cp fragment -His-tag are immobilized via an anti—His tag antibody on the surface of an ELISA plate.
- anti-vWF-cp antibodies bound to vWF-cp or vWF-cp fragment are detected by a second antibody phosphatase conjugate recognizing the constant human antibody region.
- the phosphatase reacted with an appropriate substrate resulting in a chromogenic reaction and a yellow color. The intensity of the color is measured and the amount of antibody in the sample is determined by comparison with a standard curve comprising a known amount of anti-vWF antibody.
- a commercially available, BSA free, anti—His tag antibody (“carrier-antibody”; Qiagen, Germany) is diluted to a final concentration of 2 ⁇ g/mL in PBS pH 7.4. 100 ⁇ l per well is incubated for four hours at room temperature in a 96 well-microtiter plate. After three washing steps using PBST pH 7.4 (PBS buffer containing 0.1% (v/v) Tween 20), 250 ⁇ l of a blocking solution, containing PBS pH 7.4 and 2% (w/v) bovine serum albumin, are added and incubated at 4° C. over night to block all free binding sites.
- PBST pH 7.4 PBS buffer containing 0.1% (v/v) Tween 20
- 250 ⁇ l of a blocking solution containing PBS pH 7.4 and 2% (w/v) bovine serum albumin
- vWF-cp His tag labelled preparation
- vWF-cp concentration is 1.5 ⁇ g/mL corresponding to 10 U/mL protease activity.
- vWF-cp samples are diluted to the final concentration in PBS 2% BSA. Due to the coated anti—His antibody recombinant vWFcp/his-tag is captured and immobilized via the carrier antibody. After two hours at room temperature, ten washing steps follow. The washing buffer contains PBS pH7.4 and 0.1% (v/v) Tween 20.
- Plasma samples of patients are diluted 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:600, 1:800 and 1:1200 in PBS pH 7.4 containing 2% BSA and 100 ⁇ l of each dilution is incubated at room temperature for 3 hours on the recombinant vWF-cp-containing wells.
- Inhibitory antibodies are bound on the surface of the immobilized vWF-cp and unbound antibodies are washed away by ten washing steps using PBST pH 7.4.
- Detection of human antibodies is performed with a mouse anti-human IgG Fc specific antibody or mouse anti-human IgM antibody, alkaline phosphatase conjugated.
- the antibody is diluted 1:60000 in PBS 2% BSA to the final working solution and incubated for 2 hours at room temperature (100 ⁇ l/well), followed by ten washing steps with PBST pH 7.4.
- Addition of an alkaline phosphatase substrate (PNPP) results in a yellow color, whereby the color intensity reflects the amount of bound antibody (antibody/vWF-cp).
- PNPP alkaline phosphatase substrate
- the color intensity is measured in an ELISA reader and the amount of antibody within the plasma sample is calculated in reference to a standard curve of NP by serial dilution.
- As negative control dilutions of normal human plasma (NHP) are treated accordingly.
- FIGS. 2A and 2B The results show that human anti-vWF-cp antibodies in a patients can be clearly detected in at least a plasma dilution of 1:600.
- Normal human plasma is used as control and the standard deviation (SD) calculated for several plasma lots. Antibody titres above that of normal human plasma+2 SD are evaluated as positive.
- Samples from patients with TTP and normal plasma samples are subjected to ELISA comprising immobilized vWF-cp.
- the results are shown in Table 1.
- Patient 1 has an IgG titer of 1:600 and an IgM titer of 1:400.
- the IgG titer of patient 2 is much higher (1:1200) while the IgM titer is only 1:100.
- Patient 1 suffers from an acute TTP, while patient 2 is in remission after TTP.
- Patient 1 shows no inhibitory titer, whereas patient 2 has an inhibitory titer of about 60 U/mL.
- Normal human plasma shows no reaction.
- ADAMTS-13 levels of patients #1 and #2 can be clearly detected; normal human plasma shows the same levels.
- Patient #3 is being characterized to carry a genetic defect on one allele causing a 50% reduced activity. A 50% reduction on protein amount can also be seen in our assay system.
- Patient #4 is being characterized to completely lack ADAMTS-13 protein due to a homozygous nonsense mutation. Consequently, no protein could be detected.
- GQGTPSLVPHEEAAAPGRTTATPAGASLEWSQARGLLFSPARQPRRL LPGPQENSVQSSACGRQHLEPTGTIDMRGPCQADCAVAIGR PLGEVVTLRVLESSLNCSAGDMLLLWGRLTRKMCRKLLDMTFSSK TNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWG EIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANA SYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLKAQALRG QYWTLQSWVPEMQDPQSWKGKEGT 7440 (rp) GGTTCCTTCCTTTCCCTTCCAGGACTG
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Abstract
Description
Fusion: Thioredoxin (N-terminal), 6xHis-tail (C-terminal) |
DNA-fragment | ||
(bp) | protein-fragment (aa) | region in ADAMTS13 |
88-222 | 30(P)-74(R) | Propeptid |
223-1317 | 75(A)-439(E) | Cat./Disintegr./ |
1156-1317 | 386(R)-439(E) | |
1318-2055 | 440(K)-685(A) | Cys-rich/spacer |
2056-3393 | 686(W)-1131(V) | tsp1#2-8 |
3394-4281 | 1132(G)-1427(T) | Cub1 + 2 |
TABLE 1 |
Anti-vWF-cp antibody detection ELISA. IgG as well as IgM titers of two patients. |
1:20 | 1:50 | 1:100 | 1:200 | 1:300 | 1:400 | 1:600 | 1:800 | 1:1200 | ||
|
+ + + + | + + + + | + + + | + + + | + + | + + | + | − | − |
|
+ + + | + + + | + + + | + + | + + | + | − | − | − |
NP | − | − | − | − | − | − | − | − | − |
IgG#2 | + + + + | + + + + | + + + + | + + + | + + + | + + + | + + | + + | + |
IgM#2 | + + | + + | + | − | − | − | − | − | − |
NP | − | − | − | − | − | − | − | − | − |
TABLE 2 |
Analysis of the binding on different ADAMTS-13 fragments of patient's antibodies |
Catalytic, | Catalytic, | Cys- | |||||||||
Catalytic | Catalytic | disintegrin, | disintegrin, | Cys-rich, | rich, | Tsp | Tsp | CUB | CUB | ||
domain, | domain, | tsp1 | tsp1 | spacer, | spacer, | 2-8, | 2-8, | 1 + 2 | 1 + 2 | ||
1:50 | 1:100 | 1:50 | 1:100 | 1:50 | 1:100 | 1:50 | 1:100 | 1:50 | 1:100 | ||
|
− | − | − | − | − | − | + + | + | + | − |
|
− | − | − | − | − | − | + + | + | + | − |
NP | − | − | − | − | − | − | − | − | − | − |
IgG#2 | + + + + | + + + | + + | + + | − | − | − | − | − | |
IgM#2 | + + | + + | + | − | − | − | − | − | − | |
NP | − | − | − | − | − | − | − | − | − | |
TABLE 3 |
Detection of ADAMTS-13 levels in plasma using anti-vWF-cp antibodies for capturing. |
1:20 | 1:50 | 1:100 | 1:200 | 1:300 | 1:400 | 1:600 | 1:800 | 1:1200 | ||
ADAMTS-13 | + + + + | + + + + | + + + + | + + + | + + + | + + + | + + | + | − |
#1 | |||||||||
ADAMTS-13 | + + + + | + + + + | + + + + | + + + | + + + | + + + | + + | + | − |
#2 | |||||||||
ADAMTS-13 | + + + | + + + | + + + | + + | + + | + | − | − | − |
#3 | |||||||||
ADAMTS-13 | − | − | − | − | − | − | − | − | − |
#4 | |||||||||
NP | + + + + | + + + + | + + + + | + + + | + + + | + + + | + + | + | − |
TABLE 4 | ||||
PRIMER | ADAMTS-13 | |||
(Baxter #) | DNA sequence (5′ → 3′) | DOMAIN | PRIMARY SEQUENCE | |
7442 (dp) | CCCTCCCATTTCCAGCAGAGTTGTCTT | Propeptid | SEQ ID. 1: | |
PSHFQQSCLQALEPQAVSSYLSPGAPLKGRPPSPGFQRQRQRQRR | ||||
7443 (rp) | CCGCCTCTGCCTCTGCCTCTG | |||
7359 (rp) | CTCGCAGGCCTGAGTGTTGCACATCTC | Catalytic/ | SEQ ID. 2: | |
disintegrin/ | AAGGILHLELLVAVGPDVFQAHQEDTERYVLTNLNIGAELLRDPSLG | |||
Tsp-1/#1 | AQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDD | |||
TDPGHADLVLYITRFKLEDPDGNRQVRGVTQLGGACSPTWSCLIT | ||||
EDTGFDLGCTIAHEIGHSFGLEHDGAPGSGCGPSGHVMASDGAA | ||||
PRAGLAWSPCSRRQLLSLLSAGRARCVWDPPRPQPGSAGHPPD | ||||
AQPGLYYSANEQCRVAFGPKAVACTFAREHLDMCQALSCHTDPL | ||||
DQSSCSRLLVPLLDGTECGVEKWCSKGRCRSLVELTPIAAVHG | ||||
RWSSWGPRSPCSRSCGGGVVTPPPQCNNPRPAFGGRACVGAD | ||||
LQAEMCNTQACE | ||||
7360 (dp) | GCTGCAGGCGGCATCCTACACCTG | |||
7600 (dp) | CGCTGGTCTAGCTGGGGTCCC | Tsp-1//#1 | SEQ ID. 3: | |
RWSSWGPRSPCSRSCGGGVVTPPPQCNNPRPAFGGRACVGAD | ||||
LQAEMCNTQACE | ||||
7601 (rp) | CTCGCAGGCCTGAGTGTTGCA | |||
7357 (rp) | GGCCTGCCGTGGCTTAGGCTGGAAGTA | Cystein-rich/ | SEQ ID. 4: | |
spacer | KTQLEFMSQQCARTDCQPLRSSPGGASFYHWGAAVPHSQGDAL | |||
CRHMCRAIGESFIMKRGDSFLDGTRCMPSGPREDGTLSLCVSGS | ||||
CRTFGCDCRMDSQQVWDRCQVCGGDNSTC | ||||
SPRKGSFTAGRAREYCTFLTCTPNLTSCYIANHRPLFTHLAVRIGG | ||||
RYVVAGKMSISPNTTYPSILLEDGRVEYRVALTEDRLPRLEEIRIW | ||||
GPLQEDADIQVYRRYGEEYGNLTRPDITFTYFQPKPRQA | ||||
7358 (dp) | AAGACCCAGCTGGAGTTCATGTCGCAA | |||
7441 (dp) | TGGGTGTGGGCCGCTGTGCGT | Tsp-1/ | SEQ ID. 5: | |
#2–8 | WVWAAVRGPCSVSSGAGLRWVNQSCLDQARKELVETVQCQGSQQPPA | |||
WPEACVLEPCPPYWAVGDFGPCSASCGGGLRERPVRCVEAQGSLLKT | ||||
LPPARCRAGAQQPAVALETCNPQPCPARWEVSEPSSCTSAGGAGLAL | ||||
ENETCVPGADGLEAPVTEGPGSVDEKLPAPEPCVGMSCPPGWGHLDA | ||||
TSAGEKAPSPWGSIRTGAQAAHVWTPVAGSCSVSCGRGLMELRFLCM | ||||
DSALRVPVQEELCGLASKPGSRREVCQAVPCPARWQYKLAACSVSCG | ||||
RGVVRRILYCARAHGEDDGEEILLDTQCQGLPRPEPQEACSLEPCPP | ||||
RWKVMSLGPCSASCGLGTARRSVACVQLDQGQDVEVDEACAALVRPE | ||||
ASVPCLIADCTYRWHVGWMECSVSCGDGIQRRRDTCLGPQAQAPVPA | ||||
DFCQHLPKPVTVRGCWAGPCV | ||||
7444 (rp) | CACACAGGGCCCAGCCCAGCA | |||
7439 (dp) | | Cub | 1+2 | SEQ ID. 6: |
GQGTPSLVPHEEAAAPGRTTATPAGASLEWSQARGLLFSPARQPRRL | ||||
LPGPQENSVQSSACGRQHLEPTGTIDMRGPCQADCAVAIGR | ||||
PLGEVVTLRVLESSLNCSAGDMLLLWGRLTRKMCRKLLDMTFSSK | ||||
TNTLVVRQRCGRPGGGVLLRYGSQLAPETFYRECDMQLFGPWG | ||||
EIVSPSLSPATSNAGGCRLFINVAPHARIAIHALATNMGAGTEGANA | ||||
SYILIRDTHSLRTTAFHGQQVLYWESESSQAEMEFSEGFLKAQALRG | ||||
QYWTLQSWVPEMQDPQSWKGKEGT | ||||
7440 (rp) | GGTTCCTTCCTTTCCCTTCCAGGACTG | |||
Claims (7)
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US10/422,052 US7763430B2 (en) | 2003-04-22 | 2003-04-22 | Diagnostic assay for anti-von Willebrand Factor cleaving protease (ADAMTS13) antibodies |
EP04725025A EP1616188B1 (en) | 2003-04-22 | 2004-04-01 | Diagnostic assay for anti-von willebrand factor cleaving protease (adamts13) antibodies |
AU2004232878A AU2004232878B2 (en) | 2003-04-22 | 2004-04-01 | Diagnostic assay for anti-von willebrand factor cleaving protease (ADAMTS13) antibodies |
PCT/EP2004/003483 WO2004095027A1 (en) | 2003-04-22 | 2004-04-01 | Diagnostic assay for anti-von willebrand factor cleaving protease (adamts13) antibodies |
JP2006504955A JP4542545B2 (en) | 2003-04-22 | 2004-04-01 | Diagnostic Assay for Anti-Von Bilvirant Factor Cleaving Protease (ADAMTS13) Antibody |
CA002522795A CA2522795A1 (en) | 2003-04-22 | 2004-04-01 | Diagnostic assay for anti-von willebrand factor cleaving protease (adamts13) antibodies |
ES04725025T ES2406703T3 (en) | 2003-04-22 | 2004-04-01 | Diagnostic assay for von Willebrand factor cleavage anti-protease antibodies (ADAMTS13) |
CN2004800106621A CN1777809B (en) | 2003-04-22 | 2004-04-01 | Diagnostic assay for anti-von willebrand factor cleaving protease (ADAMTS13) antibodies |
JP2009205368A JP2010025941A (en) | 2003-04-22 | 2009-09-04 | Diagnostic assay for anti-von willebrand factor cutting protease (adamts13) antibody |
US12/829,872 US7943326B2 (en) | 2003-04-22 | 2010-07-02 | Diagnostic assay for anti-von Willebrand Factor cleaving protease (ADAMTS13) antibodies |
Applications Claiming Priority (1)
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US10/422,052 US7763430B2 (en) | 2003-04-22 | 2003-04-22 | Diagnostic assay for anti-von Willebrand Factor cleaving protease (ADAMTS13) antibodies |
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US12/829,872 Division US7943326B2 (en) | 2003-04-22 | 2010-07-02 | Diagnostic assay for anti-von Willebrand Factor cleaving protease (ADAMTS13) antibodies |
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US12/829,872 Expired - Lifetime US7943326B2 (en) | 2003-04-22 | 2010-07-02 | Diagnostic assay for anti-von Willebrand Factor cleaving protease (ADAMTS13) antibodies |
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AU (1) | AU2004232878B2 (en) |
CA (1) | CA2522795A1 (en) |
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CA2550939A1 (en) * | 2003-12-22 | 2005-07-07 | Mitsubishi Kagaku Iatron, Inc. | Method of detecting thrombosis by measuring von willebrand factor-cleaving protease |
US7270976B2 (en) * | 2004-07-19 | 2007-09-18 | American Diagnostica, Inc. | Methods for measuring ADAMTS13 activity and protein on platelets and in plasma |
JP4875495B2 (en) * | 2004-11-08 | 2012-02-15 | 三菱化学メディエンス株式会社 | Method for detecting platelet thrombosis or organ damage |
EP1686176A1 (en) | 2005-01-28 | 2006-08-02 | Icon Genetics AG | Production of antibodies in plants with plus-sense single-stranded viral RNA vectors |
AU2006336468B2 (en) | 2005-02-11 | 2012-04-12 | University Of Southern California | Method of expressing proteins with disulfide bridges |
WO2006133955A1 (en) * | 2005-06-17 | 2006-12-21 | Baxter International Inc. | Adamts13-comprising compositions having thrombolytic activity |
EP1739179A1 (en) * | 2005-06-30 | 2007-01-03 | Octapharma AG | Serum-free stable transfection and production of recombinant human proteins in human cell lines |
JP5067917B2 (en) * | 2005-12-28 | 2012-11-07 | アルフレッサファーマ株式会社 | Separation and purification method of ADAMTS13 |
US20090004673A1 (en) * | 2006-01-31 | 2009-01-01 | Mitsubishi Kagaku Iatron, Inc. | Method for Determining Condition of Disseminated Intravascular Coagulation |
DE102007031708A1 (en) * | 2007-07-06 | 2009-01-08 | Dade Behring Marburg Gmbh | Determination of von Willebrand factor activity in the absence of ristocetin |
US8802394B2 (en) | 2008-11-13 | 2014-08-12 | Radu O. Minea | Method of expressing proteins with disulfide bridges with enhanced yields and activity |
AU2012255027B2 (en) * | 2011-05-19 | 2015-09-24 | Takeda Pharmaceutical Company Limited | Detection of circulating ADAMTS13-antibody complexes |
RU2641234C2 (en) * | 2011-11-20 | 2018-01-16 | Эф-Ай-Оу Корпорейшн | Method, system and device for quality control using sensors for use with devices for biological/ecological rapid diagnostic tests |
AU2013203062C1 (en) | 2013-03-15 | 2018-06-28 | Takeda Pharmaceutical Company Limited | Subcutaneous administration of adamts13 |
CN106442969B (en) * | 2016-08-23 | 2018-08-31 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of kit for detecting botulinum toxin |
CN106596936B (en) * | 2016-12-19 | 2019-05-31 | 吴江华药生物技术有限公司 | Method, detection kit and the preparation method of Quantitative in vitro measurement vWF-CP enzymatic activity |
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- 2004-04-01 CA CA002522795A patent/CA2522795A1/en not_active Abandoned
- 2004-04-01 ES ES04725025T patent/ES2406703T3/en not_active Expired - Lifetime
- 2004-04-01 WO PCT/EP2004/003483 patent/WO2004095027A1/en active Application Filing
- 2004-04-01 AU AU2004232878A patent/AU2004232878B2/en not_active Expired
- 2004-04-01 JP JP2006504955A patent/JP4542545B2/en not_active Expired - Lifetime
- 2004-04-01 CN CN2004800106621A patent/CN1777809B/en not_active Expired - Lifetime
-
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- 2009-09-04 JP JP2009205368A patent/JP2010025941A/en not_active Withdrawn
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Also Published As
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AU2004232878A1 (en) | 2004-11-04 |
US7943326B2 (en) | 2011-05-17 |
EP1616188A1 (en) | 2006-01-18 |
US20040214346A1 (en) | 2004-10-28 |
JP2006524321A (en) | 2006-10-26 |
JP2010025941A (en) | 2010-02-04 |
ES2406703T3 (en) | 2013-06-07 |
CN1777809A (en) | 2006-05-24 |
CA2522795A1 (en) | 2004-11-04 |
US20100273183A1 (en) | 2010-10-28 |
JP4542545B2 (en) | 2010-09-15 |
EP1616188B1 (en) | 2013-03-06 |
AU2004232878B2 (en) | 2009-05-28 |
CN1777809B (en) | 2013-08-21 |
WO2004095027A1 (en) | 2004-11-04 |
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